Indeed, a biochemical property exists which is specific to Candida albicans consisting in an enzymatic activity exerted by an enzyme belonging to the group comprising hexosaminidases and present in the species Candida albicans.
The determination of this enzymatic activity is the subject of a test using a culture and detection medium, marketed by Merck-Clevenot laboratories under the trademark Fluoroplate Candida Agar, comprising a nutrient base metabolized by yeasts, and a fluorigenic substrate capable of being hydrolyzed by N-acetyl-.beta.-D-galactosaminidase releasing a fluorescent product. The fluorigenic substrate is 4-methylumbelliferyl-N-acetyl-.beta.-D-galactosaminide, also termed 4-methylumbelliferyl-2-acetamido-2-deoxy-.beta.-D-galactopyranoside. Enzymatic hydrolysis of the latter by Candida albicans releases 4-methylumbelliferone, a blue fluorescent product detectable under UV radiation at 360 nm.
This test has also been described in the following document: M Manafi et al, Journal of Microbiological Methods, vol 14, No 2, 1st Aug. 1991, Amsterdam, NL, pages 103-108.
Other documents have also described, with an appropriate nutrient base, the same fluorigenic substrate or a different chromogenic substrate, capable of being hydrolyzed by an enzyme of the hexosaminidase type to release a colored or fluorescent product:
J. L. Perry et al, Journal of Clinical Microbiology, vol 12, No. 6, 1989, Washington D.C., USA, pages 2424-2425 PA1 and MT Dalton et al, Diagnostic Microbiology and Infections [sic] Disease, vol 12, No. 6, 1989, New York, pages 521-523 PA1 a) providing a detection medium comprising a nutrient base metabolized by said yeasts, a fluorogenic or chromogenic substrate capable of being hydrolyzed by an enzyme characteristic of said yeasts selected from the group consisting of hexosaminidases, to release a marker and an hexosamine portion, and an activator selected from the group consisting of hexosamine-containing compounds which are different from said substrate, and which do not inhibit the hydrolysis of said substrate by said enzyme, PA1 b) providing a sample assumed to contain said yeasts, PA1 c) contacting said sample with said detection medium, PA1 d) incubating said sample contacted with said detection medium in step c), whereby in case of the presence of said yeasts, the fluorogenic or chromogenic substrate is split into said marker and said hexosamine portion, in addition to said activator, PA1 e) observing the absence or the presence of said marker in the mixture of step d). PA1 the structural analogs of the substrate whose hexosamine portion is identical to that of the substrate and the aglycon attached to the oxygen carried by the anomeric carbon is different from the chromogenic or fluorigenic radical of the substrate, and PA1 the structural analogs of the substrate whose hexosamine portion is different from that of the substrate and the aglycon attached to the oxygen carried by anomeric carbon is identical to the chromogenic or fluorigenic radical of the substrate. PA1 peptones, from 0.01 to 40 g/l, such as meat peptone, the product marketed by the Company Biomerieux under the trademark bioSoyase or analog, or alternatively a mixture of peptones; preferably, the peptone or mixture of peptones is present in the medium at about 6 g/l.+-.0.5 g/l; PA1 a yeast extract, from 0.01 to 40 g/l, preferably about 1.5 g/l, providing vitamins for growth of the yeasts; PA1 a carbon source, such as glucose, glycerol, an acetate, a pyruvate, a lactate, arginine, an aminobutyrate, or a mixture of these components, in a proportion of 0 to 10 g/l; the carbon source is preferably glucose in an amount of 1 g/l; PA1 a buffer added to the medium in order to obtain a pH of between 5 and 8.5 which is appropriate for the development of Candida albicans; the buffer is chosen from phosphate, Tris, Hepes (N-2-hydroxyethylpiperazine-N'-2-ethasulfonic acid) and citrate buffers in a proportion of 2.5 to 100 mM; preferably, the buffer is a 10 mM phosphate buffer for adjusting the pH of the medium to a value in the region of 7; PA1 Agar, from 11 to 20 g/l, preferably 15 g/l.
have described more particularly the use of 4-methylumbelliferyl-N-acetyl-.beta.-D-galactosaminide, also termed 4-methylumbelliferyl-2-acetamido-2-deoxy-.beta.-D-galactopyranoside.
However, besides the fact that these detection media require the use of a source of UV radiation to visualize the reaction, the latter is only faintly detectable after 24 hours and is faintly detectable after 48 hours, making the test unusable within the context of rapid detection.
Furthermore, diffusion, in the medium, of the fluorescent product released by enzymatic hydrolysis reduces the probability of detecting Candida albicans in a plurimicrobial culture.
The problem of developing a method for selectively and rapidly detecting Candida albicans is therefore not yet resolved. Nevertheless, there is a great need given that Candida albicans is the species most commonly isolated from clinical samples and causes more or less severe infections of the skin, nails and mucous membranes in individuals with normal immune defenses, and very serious infections in weakened individuals and in particular those infected with the HIV virus.